Lymphedema is a lifelong disease characterized by tissue swelling, fibrosis, and increased risk of infections for millions of people in the US and is caused by impaired lymph flow. While congenital gene mutations can cause lymphedema, breast cancer surgery to remove lymph nodes is the most common cause in developed countries. Analyses of lymphatic vessels in human patients have revealed retrograde lymph flow and leaky lymphatic vessels, indicating valve dysfunction. An effective treatment for lymphedema is currently lacking, but targeted therapies to restore lymphatic valve function and prevent leakage would ideally enhance lymph flow in these patients. However, surprisingly little is known about the molecules or mechanisms regulating lymphatic vessel permeability because no methods existed to quantify lymphatic permeability. To address this gap, our lab recently developed the only approach to quantify the permeability of lymphatic vessels from knockout mice. The current proposal has combined this new physiological approach with inducible, tissue-specific knockout and transgenic mice to investigate lymphatic endothelial junction protein signaling. Our results suggest a new paradigm ? that a single junction protein is capable of regulating both valve development and lymphatic vessel permeability. The central hypothesis of this proposal is that constant signaling through the junction protein, VE- cadherin, maintains normal valve structure and lymphatic barrier function. Our findings show that VE-cadherin is required for lifelong valve maintenance by providing persistent cell signals in response to shear stress, and the same pathway prevents excessive lymphatic permeability (i.e. leakage). The central hypothesis will be tested by the following two aims investigating the role of lymphatic junction proteins: Aim 1 will test whether VE-cadherin regulates lymphatic valve formation and maintenance by transducing shear stress into intracellular signals (i.e. mechanotransduction), and Aim 2 will determine whether the same mechanotransduction signaling events regulate lymphatic permeability in the valve and non-valve areas of lymphatic collecting vessels. The completion of these aims will lead to new strategies to target VE-cadherin signaling to prevent the development of lymphedema in at-risk patients.